Glucose (carbon catabolite) repression is a major regulatory mechanism in prokaryotes and eukaryotes. The expression of many genes is severely repressed in response to glucose availability, and derepression of gene expression when glucose is scarce is an important aspect of the response to nutrient limitation. Our goal is to understand this global regulatory mechanism in molecular detail. Previously, we have identified genes required for control of the SUC2 (invertase) gene and other glucose repressible genes in the yeast Saccharomyces cerevisiae. We propose here to continue our efforts to identify key components of the regulatory pathway and to assign them biochemical functions. We will focus on the role of the SNF1 protein kinase, which is essential for release from glucose repression, and the proteins that interact with SNF1. Three genes that potentially encode activators or targets of the kinase were isolated as multicopy suppressors that compensate for reduced SNF1 kinase activity. Detailed molecular analysis is proposed to ascertain their functional relationship to SNF1. Additional genes encoding proteins that interact physically with SNF1 will be isolated by the 'two hybrid' genetic system. Mutations in SSN6 and TUP1 suppress the requirement for SNF1 and cause high-level constitutive invertase expression. Recent evidence indicates that an SSN6/TUP1 complex associates with DNA-binding proteins and represses transcription of SUC2 and other genes. To examine the mechanism of negative regulation, we win identify proteins that interact with SSN6/TUP1. Mutations in the MIG1 and SSN1 genes partially relieve glucose repression and suppress snf1, and MIG1 is a candidate for a DNA-binding protein that tethers SSN6/TUP1 to SUC2 DNA. Genetic and molecular analysis will elucidate their relationship to the SNF1 kinase and to the regulatory pathway. A grr1 mutation relieves glucose repression of many genes but makes SUC2 expression glucose inducible. Genetic analysis of GRR1 is proposed to assess its role in glucose repression, and SUC2 sequences responsible for glucose inducibility will be mapped to allow dissection of this complex response. Finally, we have isolated mutations in five new genes (SNF7-SNF10 and CID1) that affect invertase expression, and we will clone the genes to assess their function in the regulatory pathway.